Prior to the identification of specific human tumour antigens, many clinical trials were performed attempting to immunise cancer patients against either whole cancer cells or subcellular fractions from cancer cells. The identification of genes encoding tumour antigens, however, has made it possible to develop specific immunotherapies based on attacking tumour cells bearing the identified antigens. A variety of clinical approaches utilising these genes or gene products are possible as summarised in the following table.
Active immunotherapy (“Cancer vaccines”)1. Immunisation with:i) purified antigenii) immunodominant peptide (native or modified)iii) “naked” DNA encoding the antigeniv) recombinant viruses encoding the antigenv) antigen presenting cells pulsed with protein or peptide(or transfected with genes encoding the antigen)2. Use of cytokine adjuvants such as IL-2 and IL-12 administeredsystemically or encoded by the immunising vectorPassive immunotherapy (“Adoptive immunotherapy”)1. Transfer of cells sensitized in vitro to the specific antigen(bulk or cloned populations)2. Transduction of effector cells (or stem cells) with genesencoding T cell receptors that recognise specific antigens.
Immunisation with intact protein has the potential advantage of simultaneously immunising against both class I and class II epitopes but requires extensive and time-consuming efforts to purify large amounts of tumour antigen. The identification of class I and class II peptide within a tumour antigen makes it possible to immunise with high levels of pure synthetic peptide. The peptide approach also has the advantage that one can choose between a class I (cellular) and a class II type response (or mixture) by choosing which epitopes to use. Immunisation with peptide also means that subdominant and/or cryptic epitopes can be chosen (as the need for antigen processing may be bypassed or reduced to a “trimming role”) in order to stimulate a different subset of T cells. Also the peptide may be modified (for example at their HLA class I or II anchor sites) to increase their immunogenicity.
In the past few years, much attention has been given to the role of CD8+ T cells in tumour immunity. Tumour-specific CD8+ CTLs have been shown to be capable of lysing tumour cells directly and eradicating tumour masses in vivo in animal models. However, CD4+ T cells are also thought to play a critical role (Wang and Rosenberg (1999) Immunological Reviews 170:85-100) and it may be that optimal cancer vaccines require the participation of both CD4+ and CD8+ T cells.
A number of oncofoetal or tumour-associated antigens (TAAs) have been identified and characterised in human and animal tumours. In general, TAAs are antigens expressed during foetal development which are downregulated in adult cells, and are thus normally absent or present only at very low levels in adults. Tumour cells have been observed to resume expression of TAAs, and the application of TAAs for tumour diagnosis, targeting and immunotherapy has therefore been suggested.
The TAA 5T4 (see WO 89/07947) has been previously characterised. It is a 72 kDa membrane glycoprotein highly expressed on placental trophoblasts. Its expression on normal adult tissues is restricted to some specialised epithelia, but it is highly expressed and broadly distributed throughout a wide range (>75%) of carcinomas including gastric, colorectal, breast and ovarian cancer (see Table). It appears to be strongly correlated to metastasis in colorectal and gastric cancer. The full nucleic acid sequence of human 5T4 is known (Myers et al., 1994 J Biol Chem 169: 9319-24).
TABLEDistribution of Human 5T4Tumour Type5T4 Frequency (%)Breast84Ovarian71Gastric74Colorectal85    (Starzynska et al., Eur J Gastroenterol Hepatol 1998 June; 10(6):479-84; Starzynska et al., Br J Cancer 1994 May; 69(5):899-902; Starzynska et al., Br J Cancer 1992 November; 66(5):867-9)
5T4 has been proposed as a marker, with possible mechanistic involvement, for tumour progression and metastasis potential (Carsberg et al., (1996) Int J Cancer 1996 Sep. 27; 68(1):84-92). 5T4 has also been proposed for use as an immunotherapeutic agent (see WO 00/29428) and is used in TroVax® (Oxford Biomedica Ltd), a cancer vaccine in clinical development for delivery of 5T4 using an attenuated vaccinia virus vector (MVA). TroVax® is currently being evaluated in phase II clinical trials in late stage colorectal and renal cancer patients.
Cellular immune responses are directed against peptide sequences from an antigen's primary structure and are therefore less easily identified and monitored. CTL antigen receptors are only able to recognise antigens which have been processed and subsequently presented in the context of major histocompatibility complex (MHC) class I molecules on an antigen presenting or target cell. Presentation of antigens by MHC class I involves proteosome-mediated degradation of cytosolic proteins into peptides which are transported and bound to MHC class I molecules (in complex with β2-Microglobulin (β2M)) in the endoplasmic reticulum, before being translocated to the cell surface where they become available for T cell scrutiny.
Certain peptide epitopes of 5T4 that can bind specific MHCI (or MHCII) have been identified.
In addition, a number of epitope-predictive algorithms have been developed and are freely available. However, these are predominantly based on published data relating to the most common allele in the Caucasian population, HLA A*0201. Furthermore, while these are useful, such algorithms can produce high levels of false positive and negative results.
Accordingly there is a need for the identification of additional 5T4 epitopes and, in particular, there is a need for an increased repertoire of epitopes that can bind to a broader range of MHCI or MHCII.
This need is particularly relevant to 5T4. This is because 5T4 is a self-antigen meaning that the greatest challenge associated with mounting an effective anti-tumour immune response is the breaking of immunological tolerance. The magnitude of immunological responses induced against a self-antigen are usually lower than those observed against foreign pathogens such as viruses. This means that effective and sensitive monitoring of a broad range of immunological responses is essential in order to prove efficacy in breaking tolerance and inducing immune responses. Since cytotoxic T cells are thought to be a key mediator of tumour cell killing, the ability to detect and characterise 5T4 specific CTL responses is essential making the need for additional peptide epitopes of 5T4 particularly acute.
Additional epitopes will provide diagnostic tools allowing routine monitoring of clinical immune responses to become more focussed, streamlined, and sensitive and enable more robust assessments of possible correlations between 5T4-specific immune responses and clinical benefit.